Method For Synthesizing Single-Stranded Nucleic Acid

ABSTRACT

This invention relates to a method for selectively and efficiently synthesizing one of the sense or antisense strands of double-stranded nucleic acid. The method for synthesizing single-stranded nucleic acid according to this invention comprises the following steps of: 1) cleaving, with a restriction enzyme, double-stranded DNA having a restriction enzyme recognition sequence at a portion closer to the 5′ side than the target sequence in such a manner that a) a fragment having an overhanging 3′ terminus is formed, and b) base sequences of the single-stranded regions of the fragments after the cleavage differ from each other; 2) to the single-stranded region of the DNA fragment that was cleaved with the restriction enzyme, annealing a primer having a base sequence complementary to the region on at least its 3′ terminus; and 3) synthesizing a nucleic acid by a strand displacement-type polymerase starting from the 3′ terminus of the primer. Further, a single-stranded nucleic acid can be more efficiently synthesized by amplifying the double-stranded DNA having the restriction enzyme recognition sequence by the LAMP reaction.

TECHNICAL FIELD

The present invention relates to a method for synthesizingsingle-stranded DNA, and more particularly to a method for selectivelysynthesizing one of the sense or antisense strands of double-strandedDNA.

Background Art

The most common method for preparing single-stranded DNA that is used asa probe in the hybridization assay is carried out by denaturingdouble-stranded DNA by heat or alkali. With this method, however,complementary strands are present in the product, and thus,complementary strands are rebound to each other under conditions forforming a double strand. This sometimes deteriorates the hybridizationefficiency. Also known is a method for preparing single-stranded DNAthrough cloning by utilizing a single-stranded phage such as M13,although this method is costly and time-consuming.

A short single-stranded DNA sequence comprising approximately severaldozen bases can be chemically synthesized without difficulty. Incontrast, if a long single-stranded DNA sequence having more than 100bases is chemically synthesized, the yield and the accuracy of basesequences deteriorate. Although the T7 RNA polymerase can synthesizesingle-stranded RNA using DNA as a template, the template DNA needs apromoter sequence which is recognized by the aforementioned enzyme.

In contrast, WO 99/09211 discloses a method for amplifying a targetsequence on the first strand of a double-stranded nucleic acid. In thismethod, the double-stranded nucleic acid is cleaved using a restrictionenzyme, and with this cleavage, the 5′ terminus of the target sequenceon the first strand is cleaved so as to form an overhanging 3′ terminalregion on the second strand. The extension reaction is then carried outusing a primer complementary to the 3′ terminus of the second strand anda strand displacement-type polymerase and employing the second strand asa template, thereby amplifying the target sequence. In this method, arestriction site is also regenerated simultaneously with the synthesisof the target region by a primer in principle. However, the targetregion should have the restriction site at the 5′ terminus when thereaction is initiated, and the provision of the recognition site is notsufficiently disclosed. Also, this method is an improved version of theStrand Displacement Amplification (SDA) method [Proc. Natl. Acad. Sci.USA, 89, 392-396; 1992] [Nucleic Acid. Res., 20, 1691-1696; 1992], whichoriginally aimed at amplifying double-stranded DNA. Accordingly, whenthe sequences of the overhanging portions of each of the 3′ sidescreated by cleavage are identical to each other (palindrome sequences),both the sense and antisense strands are synthesized, even with the useof only one primer. Specifically, this method does not always providesingle-stranded DNA.

The SDA method is briefly described. The SDA method is a methodutilizing a special DNA polymerase. When complementary strand issynthesized starting from a primer that is complementary to the 3′ sideof a certain base sequence and a double-stranded region is present onthe 5′ side, the complementary strand is synthesized so as to displaceone strand of the double-stranded region. In the SDA method, previousinsertion of the restriction enzyme recognition sequence in the annealedsequence by a primer can allow the omission of the step of temperaturevariation that is essential in PCR. Specifically, the nick, which isgenerated by a restriction enzyme, imparts the 3′-OH group as a startingpoint of complementary strand synthesis, and strand displacementsynthesis is performed therefrom. Thus, the previously synthesizedcomplementary strand is freed as a single-strand and reused as atemplate for the next complementary strand synthesis. In this manner,the SDA method eliminated the need for complicated temperature control,which was required in PCR.

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide a method forselectively and efficiently synthesizing a relatively long sense orantisense strand.

The present inventors have conducted concentrated studies, and as aresult, they found that a single-stranded nucleic acid could beselectively and efficiently synthesized. This involves the use of amethod for amplifying DNA utilizing the LAMP method and a “restrictionenzyme which is capable of forming a fragment having an overhanging 3′terminus and cleaving so as to make base sequences of thesingle-stranded regions of the fragments after the cleavage differentfrom each other.” This led to the completion of the present invention.More specifically, the present invention comprises the following.

(1) A method for synthesizing a single-stranded nucleic acid comprisingthe following steps of:

1) cleaving, with a restriction enzyme, double-stranded DNA having arestriction enzyme recognition sequence at a portion closer to the 5′side than the target sequence in such a manner that

-   -   -   a) a fragment having an overhanging 3′ terminus is formed,            and        -   b) base sequences of the single-stranded regions of the            fragments after the cleavage differ from each other;

2) to a single-stranded region of the DNA fragment that was cleaved withthe restriction enzyme, annealing a primer having a base sequencecomplementary to the region on at least its 3′ terminus; and

3) synthesizing a nucleic acid by a strand displacement-type polymerasestarting from the 3′ terminus of the primer.

(2) The method according to (1) above, wherein the double-stranded DNAhaving the restriction enzyme recognition sequence is provided by amethod comprising the following steps of:

1) cloning the DNA to be amplified using a vector having the restrictionenzyme recognition sequence in the cloning site or adjacent to thecloning site; and

2) amplifying the region containing the restriction enzyme recognitionsequence and the DNA to be amplified by the DNA amplification methodusing a primer that anneals on the restriction enzyme recognitionsequence or the 3′ side thereof.

(3) The method according to (2) above, wherein the DNA amplificationmethod is the LAMP method.

(4) The method according to (1) above, wherein the double-stranded DNAhaving the restriction enzyme recognition sequence is provided by theDNA amplification method using a primer containing the restrictionenzyme recognition sequence.

(5) The method according to (4) above, wherein the DNA amplificationmethod is the LAMP method using the primers described in the followingA) and B):

when a first arbitrary sequence F1c and a second arbitrary sequence F2care selected in that order from the 3′ terminus of the target sequenceon the first DNA strand of the double-stranded DNA toward the 3′terminus on the DNA strand, and a third arbitrary sequence R1 and afourth arbitrary sequence R2 are selected in that order from the 5′terminus of the target sequence toward the 5′ terminus on the DNAstrand,

A) a primer containing sequence F2, which is complementary to F2c, andthe same sequence as F1c in that order from the 3′ side toward the 5′side or a primer containing sequence F2, which is complementary to F2c,the restriction enzyme recognition sequence, and the same sequence asF1c in that order from the 3′ side toward the 5′ side; and

B) a primer containing the same sequence as R2, the restriction enzymerecognition sequence, and sequence R1c, which is complementary to R1, inthat order from the 3′ side toward the 5′ side.

(6) The method according to (5) above, wherein the step of synthesizingDNA comprises the use of an outer primer that anneals to the portioncloser to the 3′ side than the inner primer.

(7) The method according to any one of (1) to (6) above, wherein thesingle-stranded region at the cleavage site created by the restrictionenzyme comprises at least 5 bases.

(8) The method according to any one of (1) to (6) above, wherein thesingle-stranded region at the cleavage site created by the restrictionenzyme comprises at least 7 bases.

(9) The method according to any one of (1) to (8) above, wherein theprimer is bound to or modified to be bindable to a detectable labelsubstance or solid phase.

(10) An inner primer pair comprising the following A) and B):

when a first arbitrary sequence F1c and a second arbitrary sequence F2care selected in that order from the 3′ terminus of the target sequenceon the first DNA strand of the double-stranded DNA toward the 3′terminus on the DNA strand and a third arbitrary sequence R1 and afourth arbitrary sequence R2 are selected in that order from the 5′terminus of the target sequence toward the 5′ terminus on the DNAstrand,

A) a primer containing sequence F2, which is complementary to F2c, andthe same sequence as F1c in that order from the 3′ side toward the 5′side, or a primer containing sequence F2, which is complementary to F2c,a recognition sequence of the following restriction enzyme, and the samesequences as F1c in that order from the 3′ side toward the 5′ side,wherein the restriction enzyme is capable of

-   -   a) forming a fragment having an overhanging 3′ terminus, and    -   b) cleaving so as to make base sequences of the single-stranded        regions of the fragments after the cleavage different from each        other; and

B) a primer containing the same sequence as R2, a recognition sequenceof the restriction enzyme, and sequence R1c, which is complementary toR1, in that order from the 3′ side toward the 5′ side.

(11) A vector for synthesizing a single-stranded nucleic acid comprisingthe following base sequence in the cloning site or adjacent to thecloning site, wherein the base sequence is cleaved with a restrictionenzyme in such a manner that

a) a fragment with an overhanging 3′ terminus is formed, and

b) base sequences of the single-stranded regions of the fragments afterthe cleavage differ from each other.

(12) A reagent kit for synthesizing a single-stranded nucleic acidcomprising at least the following reagents:

1) a restriction enzyme capable of:

-   -   a) forming a fragment having an overhanging 3′ terminus, and    -   b) cleaving so as to make the base sequences of fragments        different from each other;

2) a primer capable of annealing to the single-stranded region of theDNA fragment cleaved with the restriction enzyme;

3) a strand displacement-type polymerase; and

4) the inner primer according to (10) above or the vector forsynthesizing a single-stranded nucleic acid according to (11) above.

In the method for synthesizing a single-stranded nucleic acid accordingto the present invention, the term “single-stranded nucleic acid” refersto DNA which consists of either one of the sense or antisense strands ofthe double-stranded DNA, i.e., single-stranded DNA. The term “annealing”refers to the formation by a nucleic acid of a double-stranded structureby base pairing in accordance with the law of Watson-Crick. Accordingly,if a single-stranded nucleic acid foims such base pairing in a molecule,it is “annealing.” In the present invention, “annealing” and“hybridization” are synonymous with each other in that a nucleic acidhas a double-stranded structure formed by base pairing.

In the present invention, the term “target sequence (or region)” refersto a base sequence (or region) which should be synthesized as asingle-stranded nucleic acid. Of the template double-stranded DNA, thestrand containing the target sequence refers to the first strand and thestrand complementary thereto refers to the second strand.

Techniques according to the LAMP method are applied in the presentinvention. The LAMP method is a method for amplifying a nucleic acid,which was developed by the present inventors. In this method, an innerprimer pair or two or four specific primers prepared by adding an outerprimer pair to the inner primer pair, a strand displacement-type DNApolymerase, and a substrate nucleotide are used to rapidly andcost-effectively amplify DNA or RNA under isothermal conditions (atapproximately 65° C.). The LAMP method is described in detail in, forexample, Nagamine et al., Clinical Chemistry (2001), Vol. 47, No. 9,1742-1743, Notomi et al., Nucleic Acids Research (2000), Vol. 28, e63,and International Application (e.g., WO 00/28082, WO 01/34838, and WO01/34790). These descriptions are incorporated herein by reference.

The present invention utilizes a restriction enzyme, which is capable ofa) forming a fragment having an overhanging 3′ terminus, and b) cleavingso as to make base sequences of the single-stranded regions of thefragments after the cleavage different from each other. After thecleavage, most restriction enzymes form fragments having overhanging 5′terminuses or fragments having blunt terminuses. However, a minor numberof restriction enzymes that form fragments having the overhanging 3′terminuses after the cleavage are known. A primer is annealed to thesingle-stranded region of the DNA fragment which was cleaved using suchenzymes, and a nucleic acid can be synthesized by a polymerase. In orderto conduct the synthesis reaction of a nucleic acid by annealing aprimer to the overhanging 3′ terminal region, the single-stranded regionshould comprise at least 5 bases, and preferably at least 7 bases whenthe specificity of the base sequence and the binding strength are takeninto consideration.

Some restriction enzymes have cleavage sites in an arbitrary sequencesandwiched between recognition sites or adjacent to a recognition site.For example, BglI cleaves between the base 7 and the base 8 in thesequence GCCNNNNNGGC. The fragment cleaved with such a restrictionenzyme produces an asymmetric fragment having different base sequencesin the cleavage sites. Specifically, when the sequence GCCAAAAAGGC iscleaved with BglI, the base sequences of the cleavage site are GCCAAAAand GCCTTTT. In such asymmetric fragments, a primer (probe) having abase sequence complementary to one of them cannot bind to the other.

One example of a restriction enzyme having both characteristicsmentioned above (hereinafter it is referred to as “the restrictionenzyme of the present invention”) is TspRI. TspRI recognizes the basesequence NNCA (C or G) TGNN and cleaves between an arbitrary base at the3′ terminus and a base adjacent thereto. Thus, the single-strandedregion after the cleavage comprises 9 bases.

The principle of the method for synthesizing a single-stranded nucleicacid according to the present invention is described using TspRI as anexample. As mentioned above, TspRI is a restriction enzyme that has anoverhanging terminus of 9 bases at the cleavage surface on the 3′ side.Further, 2 bases (NN) at the both terminuses of the recognition sequenceNNCA (C/G) TGNN may be any of A, T, G, or C, and an asymmetricrecognition sequence can be formed. Utilization thereof enables thestrand-specific DNA synthesis. For example, a sequence having TspRIsites at the both ends of the BamHI cloning site is prepared. Digestionthereof with TspRI can yield four 3′-overhanging terminuses havingdifferent base sequences. Subsequently, a primer (GACACTGGA) is preparedfrom a sequence which recognizes one of the four terminuses (FIG. 1[1]). This primer is not a sequence that completely matches [2], [3], or[4], and thus, cannot be annealed. Accordingly, when the primer isannealed to [1] and causes the extension reaction, the synthesisreaction takes place using only the same strand as a template.

The techniques of the LAMP method mentioned above (Nucleic AcidsResearch (2000), Vol. 28, e63) can be applied to the above synthesisreaction. For example,

1) DNA to be amplified is cloned using a vector having the restrictionenzyme recognition sequence in a cloning site or adjacent to the cloningsite;

2) subsequently, a primer for LAMP to be annealed on the restrictionenzyme recognition sequence or the 3′ side therefrom is designed; and

3) the primer and the strand displacement-type polymerase are used toamplify by the LAMP method the region containing the restriction enzymerecognition sequence and DNA to be amplified.

Processes for synthesizing single-stranded DNA from double-stranded DNAhaving the amplified restriction enzyme recognition sequence are asdescribed above. Since the LAMP method can provide a large amount of DNAunder isothermal conditions in a short period of time, morestrand-specific single-stranded DNA can be efficiently obtained bycombining the LAMP method with the above synthesis method.

The primer for LAMP refers to a specific primer, which is used in theLAMP method, and can be easily prepared based on, for example, theaforementioned Nagamine et al., “Clinical Chemistry (2001).”

A primer, which is used for synthesizing the single-stranded DNA, is notparticularly limited as long as it complementarily binds to theoverhanging single-stranded portion at the 3′ terminus existing on thesecond DNA strand, which was cleaved with a restriction enzyme. Thelength is not necessarily completely consistent with that of thesingle-stranded region. It may be shorter than the single-strandedregion on the 5′ side or the 3′ side, or longer on the 5′ side, as faras the specificity of complementary binding is not deteriorated.

A primer may be bound to, or modified to be bindable to, a detectablelabel substance or solid phase. When labeling the primer forsynthesizing single-stranded DNA, known substances and methods forlabeling can be employed. Examples of label substances includeradioactive substances, fluorescent substances, biotins, and enzymes.These label substances can be added to a primer in accordance with aknown method, or a previously labeled nucleotide can be incorporated atthe time of chemical synthesis of a primer to prepare a label primer. Asuitable functional group may be introduced in the primer so as to bebindable to the aforementioned label substances or latex particles,magnetic particles, or the inner wall of a reaction vessel.

The label site of the primer has to be selected in such a manner thatannealing to a complementary strand or a subsequent extension reactionis not inhibited. Thus, for example, a label at the 3′ terminus is notpreferable. Depending on their molecular weight, label substances can bebound through a base sequence as a linker on the 5′ side to preventsteric hindrance from occurring.

In the method for synthesizing a single-stranded nucleic acid accordingto the present invention, the recognition sequence can be inserted intothe double-stranded DNA that does not contain a restriction enzymerecognition sequence of the present invention by conventional methodsfor gene amplification such as PCR and LCR. For example, the restrictionenzyme recognition sequence of the present invention is incorporatedinto a primer to be used, and the target sequence or a sequencecomplementary thereto is amplified. Thus, the restriction enzymerecognition sequence of interest can be inserted on the 3′ side of thetarget sequence.

As mentioned above, a vector, which comprises the restriction enzymerecognition sequence of the present invention incorporated in thecloning site of the cloning vector or adjacent to the cloning site, maybe used.

According to a preferred embodiment of the present invention, therestriction enzyme recognition sequence can be introduced intodouble-stranded DNA that does not contain a restriction enzymerecognition sequence of the present invention by the LAMP method.Specifically, a large amount of template DNA strands for synthesizing asingle-stranded nucleic acid having a restriction enzyme recognitionsequence can be rapidly prepared by performing LAMP amplification usinga primer comprising the restriction enzyme recognition sequence insertedtherein.

The restriction enzyme recognition sequences should be inserted on the5′ side of the target sequence on the first strand. Particularly, it ismore preferable if the sequences are inserted into both the 3′ side andthe 5′ side. More specifically, the restriction enzyme recognitionsequence is always necessary for the primer which is annealed to theoverhanging 3′ portion on the second strand, and it is more preferableif the sequences are contained in both the primers. This is because thepresence of restriction sites on both sides allows cleavage of bothsides of the target region by one type of enzyme and can decrease thenecessary number of enzymes. It should be noted that the base sequencesof the single-stranded regions of the fragments, which are obtained bycleaving the extension product from each primer with the restrictionenzyme, are preferably different from each other.

In the present invention, the above two types of primers are referred toas “inner primers.”

More specifically, the method for introducing a restriction enzymerecognition sequence into double-stranded DNA by the LAMP methodcomprises the following steps of:

1) selecting a first arbitrary sequence F1c and a second arbitrarysequence F2c in that order from the 3′ terminus of the target sequenceon the first DNA strand of the double-stranded DNA toward the 3′terminus on the DNA strand and selecting a third arbitrary sequence R1and a fourth arbitrary sequence R2 in that order from the 5′ terminus ofthe target sequence toward the 5′ terminus on the DNA strand;

2) preparing the following inner primers:

-   -   a) a primer containing sequence F2, which is complementary to        F2c, and the same sequence as F1c in that order from the 3′ side        toward the 5′ side, or a primer containing sequence F2, which is        complementary to F2c, the restriction enzyme recognition        sequence, and the same sequences as sequence F1c in that order        from the 3′ side toward the 5′ side; and

b) a primer containing the same sequence as R2, the restriction enzymerecognition sequence, and sequence R1c, which is complementary to R1, inthat order from the 3′ side toward the 5′ side; and

3) synthesizing DNA using each strand of the double-stranded DNA as atemplate and using the primer and the strand displacement-typepolymerase.

In the aforementioned inner primers, F2 may overlap with the restrictionenzyme recognition sequence, and the restriction enzyme recognitionsequence may overlap with F1c, or several base sequences may be insertedtherebetween. Similarly, R2 may overlap with the restriction enzymerecognition sequence, and the restriction enzyme recognition sequencemay overlap with R1c, or several base sequences may be insertedtherebetween. Preferably, distance between F2/R2 and the restrictionenzyme recognition sequence or distance between the restriction enzymerecognition sequence and F1c/R1c is 1 to 20 bases, and more preferably 1to 10 bases.

The first annealing of the inner primer to double-stranded DNA can becarried out by dissociating a part or the entire double-stranded DNA byconventional methods such as by heat or alkali. Thereafter, DNAsynthesis is repeated in the presence of a strand displacement-type DNApolymerase and a substrate nucleotide under isothermal conditions ofapproximately 55° C. to 70° C.

In the aforementioned LAMP method, two types of primers that differ fromthe inner primers can be further used. These primers anneals to theouter side, i.e., closer to the 3′ side, than the inner primers on thetemplate DNA, and they are referred to as “outer primers” in the presentinvention. The outer primers can be prepared in the following manner.

On the first DNA strand, an arbitrary sequence F3c, which is locatedcloser to the 3′ side than F2c, and an arbitrary sequence R3, which islocated closer to the 5′ side than R2, are selected, and

a) a primer containing sequence F3, which is complementary to F3c, and

b) a primer containing a sequence that is identical to R3, arerespectively prepared as the outer primers.

The DNA synthesis from the outer primer should be initiated after theDNA synthesis from the inner primer. Examples of methods for realizingthis include: a method in which the concentration of the inner primer isset higher than that of the outer primer (e.g., 2 to 50 times,preferably 4 to 25 times) (a); and a method in which the meltingtemperature (Tm) of the inner primer is set higher than that of theouter primer (b). In addition, conditions for carrying out the LAMPmethod can be suitably determined with reference to the literature orpatent mentioned above.

The LAMP method can be more efficiently carried out with the use of aprimer capable of specifically annealing to the loop portion of theamplification product having a hairpin structure formed byamplification. In the present invention, the loop-specific primer isreferred to as a “loop primer.” If the restriction enzyme recognitionsequence of the present invention is inserted into one of the 3′terminuses of the loop primer, the loop primer per se can function as aprimer for synthesizing a single-stranded nucleic acid.

The present invention also provides a reagent kit for synthesizing asingle-stranded nucleic acid for carrying out the method according tothe present invention. This kit can be used, for example, as a reagentkit for synthesizing a single-stranded nucleic acid for thehybridization assay of a particular sequence.

The aforementioned kit comprises at least the restriction enzymeaccording to the present invention, a primer capable of annealing to thesingle-stranded region of the DNA fragment cleaved with the restrictionenzyme, a strand displacement-type polymerase, and the inner primeraccording to the present invention. The kit may further comprise theouter primer and/or the loop primer according to the present invention.

The kit may further comprise, in addition to the aforementioned reagent,other reagents necessary for carrying out the synthesis method of thepresent invention, for example, a substrate nucleotide, a buffer, or amelting temperature regulator.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the principle for synthesizing the single-stranded nucleicacid using TspRI.

FIG. 2 is a photograph showing the result of the hybridization assay onthe extension product in Example 1 (A: sense oligo, B: antisense oligo).

FIG. 3 is a photograph showing the result of electrophoresis on the LAMPreaction product in Example 2 and the TspRI-treated LAMP reactionproduct (M: size marker, 1: LAMP product, 2: TspRI-treated LAMPproduct).

FIG. 4 is a photograph showing the result of detecting the primerextension product (DIG-labeled) in Example 2 (1: negative control, 2:primer extension product).

FIG. 5 is a photograph showing the result of dot blot hybridization ofthe primer extension product in Example 2.

FIG. 6 is a photograph showing the result of detecting the primerextension product (DIG-labeled) in Example 3 (1: Klenow(−), 2:Klenow(+), 3: Bst(−) 60° C., 4: Bst(+) 60° C., 5: Bst(−) 65° C., 6:Bst(+) 65° C.).

FIG. 7 is a photograph showing the result of electrophoresis on thelambda exonuclease-treated primer extension product in Example 4 (M:size marker, 1: λ(−), 2: λ(+)).

FIG. 8 is a photograph showing the result of detecting the primerextension product (DIG-labeled) in Example 5.

FIG. 9 is a photograph showing the result of dot blot hybridization ofthe primer extension product in Example 5.

FIG. 10 is a photograph showing the result of electrophoresis on theLAMP reaction product in Example 6 and the TspRI-treated LAMP reactionproduct (M: size marker, 1: LAMP product, 2: TspRI-treated LAMPproduct).

FIG. 11 is a photograph showing the result of dot blot hybridization ofthe primer extension product in Example 6.

This specification includes part or all of the contents as disclosed inthe description of Japanese Patent Application No. 2000-328219, which isa priority document of the present application.

PREFERRED EMBODIMENTS OF THE PRESENT INVENTION EXAMPLE 1

1. Preparation of Vector

PCR was carried out using the following primers, and the pBluescript IIvector (Genbank T7TspRI (Forward): 5′-GACAGTGTCGCCCTATAGTGAGTCGTATTA-3′(SEQ ID NO: 1) T3TspRI (Reverse): 5′-GGATCCAGTGTCCCTTTAGTGAGGGTTAAT-3′(SEQ ID NO: 2)

To the 40 μl of reaction system were added 1× buffer (16 mM (NH₄)₂SO₄,50 mM Tris-HCl pH 9.2, 1.75 mM MgCl₂, 0.001% (w/v) gelatin), 0.2 mMdNTPs, 1.4 U Taq DNA polymerase, Pwo DNA polymerase (EXpand LongTemplate PCR System, Roche), and 0.5 mM specific primer pair mentionedabove. PCR was repeated fifteen times under conditions of at 94° C. for20 seconds, at 62° C. for 30 seconds, and at 68° C. at 120 seconds.

The amplification product was blunt-ended using the T4 DNA polymerase,and ligation was carried out using the T4 DNA ligase. The ligationproduct was introduced into Escherichia coli DH5α, and a vector ofinterest was obtained from the resulting transformant (designated as“pBSTspRI”).

A multiple cloning site from SacI to KpnI (underlined portion) in thepBluescript II vector has been deleted from the vector obtained justabove. Instead, the vector has the TspRI-BamHI-TspRI site, and caninsert the foreign gene into the BamHI site.

(A) pBluescript II Vector:5′-GAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACAC (SEQ ID NO:10)    TTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAG   GAAACAGCTATGACCATGATTACGCCAAGCGCGCAATTAACCCTCACTAAAGGGAAC   AAAAGCTGGAGCTCCACCGCGGTGGCGGCCGCTCTAGAACTAGTGGATCCCCCGGG   CTGCAGGAATTCGATATCAAGCTTATCGATACCGTCGACCTCGAGGGGGGGCCCGGT   ACCCAATTCGCCCTATAGTGAGTCGTATTACGCGCGCTCACTGGCCGTCGTTTTACAA   CGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCC   CCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCG-3′The underlined sequence (SEQ ID NO: 11) indicates a multiple cloningsite.

(B) Sequence having the TspRI-BamHI-TspRI Site:5′-GGGACACTGGATCCGACAGTGTCGCCC-3′ (SEQ ID NO: 3)2. Cloning of DNA

pUC19 vector (GenBank DB No.: L09137) was digested with Sau3AI, and 109bp and 82 bp DNA fragments were isolated. These fragments were insertedinto the BamHI site of the vector and cloned.

109 bp Sau3AI Fragment:5′-GATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTT (SEQ IDNO: 12) GGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATC-3′

82 bp Sau3AI fragment:5′-GATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTT (SEQ ID NO:13) GGTCATGAGATTATCAAAAAGGATC-3′3. LAMP Reaction

The target region was amplified by the LAMP reaction (see Nucleic AcidsResearch (2000), Vol. 28, e63) using the vector comprising the Sau3AIfragment incorporated therein as a template. The composition of thereaction solution used is shown in Table 1, and the sequence and theconcentration of the primer are shown below. TABLE 1 Composition ofreaction solution (in 25 μL) 20 mM Tris-HCl pH 8.8 10 mM KCl 10 mM(NH₄)₂SO₄ 8 mM MgSO₄ 0.8 M Betaine 0.1% Triton X-100 5.6 mM dNTPs 8 UBst DNA polymerase (NEW ENGLAND BioLabs)Primer:Inner Primer (1600 nM)

Forward: 5′-TCCAGTGTCCCTTTAGTGAGGGTTAATTTCACACAGGAAACAGCTATGACCA-3′ (SEQID NO: 4)

Reverse: 5′-GACAGTGTCGCCCTATAGTGAGTCGTATTACCAGTCACGACGTTGTAAAACGA-3′(SEQ ID NO: 5)

Outer Primer (400 nM) Forward: 5′-GTAACGCCAGGGTTTTCC-3′ (SEQ ID NO: 6)Reverse: 5′-GAATTGTGAGCGGATAACAAT-3′ (SEQ ID NO: 7)

Loop Primer (800 nM) Forward: 5′-GCGCGCTTGGCGTAATCA-3′ (SEQ ID NO: 8)Reverse: 5′-GCGCGCTCACTGGCCG-3′ (SEQ ID NO: 9)

Target DNA, which is not heat-denatured, was prepared, and the reactionsolution was allowed to react at 65° C. The product was eluted to 200 μlof Tris-HCl (pH 8.0) using the PCR Purification Kit (QIAGEN).

3. Digestion with TspRI

The LAMP product (25 μl) was allowed to react in 50 U of TspRI (NEWENGLAND BioLabs) at 65° C. for 90 minutes. After the reaction, theproduct was eluted to 100 μl of Tris-HCl (pH 8.0) using the PCRPurification Kit (QIAGEN).

4. Primer Extension Reaction

A primer labeled with 5′-digoxigenin (DIG) (BSTspRI: 5′-GACACTGGA-3′)was added to the TspRI fragment, and the extension reaction using DNApolymerase Klenow fragment, 3′→5′exo- (NEW ENGLAND BioLabs) was carriedout. The composition of the reaction solution used is shown in Table 2.TABLE 2 Composition of reaction solution 10 mM Tris-HCl (pH 7.5) 5 mMMgCl₂ 7.5 mM Dithiothreitol 0.25 mM dNTPs 5 μM Primer (BSTspRI primer) 1U Klenow fragment, 3′ → 5′exo-(NEW ENGLAND BioLabs)

In this case, the primer BSTspRI can be annealed to only one cohesiveterminus of the TspRI-digested fragment, and thus, there should be asingle DNA strand obtained by the extension reaction.

5. Dot Blot Hybridization

A sense oligo (109A oligo) or antisense oligo (109B oligo), which wasdesigned from the base sequence of the 109 bp Sau3AI DNA fragment, wasblotted on a nylon membrane filter (BiodyneB, Pall). Sequences are shownbelow. (SEQ ID NO: 15) 109A oligo: 5′-GTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGC-3′ (SEQ ID NO: 16) 109Boligo: 5′- GCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGAC-3′

This filter was subjected to hybridization using the extension productas a probe. Hybridization was carried out using PerfectHyb buffer(TOYOBO) at 60° C. overnight. Signals were detected using the DIGNucleic Acid Detection Kit (Roche). As a result, a signal was detectedonly on the portion where the antisense oligo had been blotted (FIG. 2).This indicates that one strand-specific single-strand DNA was obtained.

EXAMPLE 2

The aforementioned 82 bp cloning product was used to conduct theexperiment in order to confirm whether or not a similar effect could beattained with another template.

The 82 bp cloning vector that was prepared in Example 1 was used as atemplate to perform the LAMP reaction under the same conditions asabove. In this LAMP reaction, the following inner primers and the sameouter primers as used in Example 1 (the same concentration as inExample 1) were used instead of the loop primer.

Inner Primer (1600 nM) (SEQ ID NO: 17) Forward:5′-GTGTCCCTTTAGTGAGGGTTAATTTCACACAGGAAACAGCTATG-3′ (SEQ ID NO: 18)Reverse: 5′-TGTCGCCCTATAGTGAGTCGTATTACCAGTCACGACGTTGTAAA-3′

The LAMP reaction product was treated with TspRI and confirmed by 2%agarose gel electrophoresis (FIG. 3).

After the confirmation of the complete digestion with TspRI byelectrophoresis, primer extension was carried out in the same manner asin Example 1. In order to confirm the implementation of primerextension, the product was electrophoresed in 2% agarose gel andtransferred to the nylon membrane (BiodyneB, Pall). Whether or not thismembrane contained the label substance in the electrophoresis productwas detected using the DIG Nucleic Acid Detection Kit (Roche) (FIG. 4).As a result, it was confirmed that the extension product from theDIG-labeled primer was contained in the TspRI product.

Since the product was confirmed as being labeled, dot blot hybridizationwas carried out using this product as a probe (FIG. 5). The blotted DNAsare as follows.

1: 82 bp antisense oligo DNA (SEQ ID NO: 14)

2: 82 bp sense oligo DNA (SEQ ID NO: 13)

3: 109A oligo

4: 109B oligo

5: heat-denatured product of 82 bp LAMP product

6: heat-denatured product of unrelated LAMP product (λ DNA amplificationproduct)

As a result, signals were found only in 1 and 5. This indicates that,even with a different template, only the strand-specific DNA wassynthesized as with Example 1. Since a signal was also found in theheat-denatured product of the LAMP product, it was found that the LAMPproduct per se could be a target of the probe. Due to its structure,i.e., the presence of the repeated sequence, the LAMP product wasconsidered to be very poor in the hybridization efficiency. In thisstudy, since hybridization was carried out with the LAMP product, thepossibility of the use of the LAMP product by spotting on the substrateof the DNA chip was suggested.

A primer, which was DIG-labeled at its 5′ terminus, was used in thisstudy. If an amino linker or a biotin-labeled primer is used, thelabeled DNA strand can be isolated.

EXAMPLE 3 Extension Reaction Using Bst DNA Polymerase

The Bst DNA polymerase, i.e., a strand displacement-type DNA synthetase,was used to determine whether or not the primer extension reaction couldbe carried out. The composition of the reaction solution used is shownin Table 3. TABLE 3 Composition of reaction solution 20 mM Tris-HCl pH8.8 10 mM KCl 10 mM (NH₄)₂SO₄ 4 mM MgSO₄ 0.1% Triton X-100 1.6 mM dNTPs1 U Bst DNA polymerase (NEW ENGLAND BioLabs)

After the reaction at 60° C. or 65° C. for 1 hour, the product waselectrophoresed in 2% agarose gel and transferred to a nylon membrane(BiodyneB, Pall) in order to confirm the implementation of primerextension. Whether or not this membrane contained the label substance inthe electrophoresis product was detected using the DIG Nucleic AcidDetection Kit (Roche) (FIG. 6). As a result, the occurrence of extensionfrom the DIG-labeled primer with the use of the Bst DNA polymerase wasconfirmed.

EXAMPLE 4 Lambda Exonuclease Treatment of Primer Extension Product

After the primer extension, free single-stranded DNA and double-strandedDNA are generated. The strand extended from the primer of thedouble-stranded DNA does not have a phosphoric acid group at its 5′terminus. Thus, if the strand is subjected to lambda exonucleasetreatment, DNA decomposition starts from the 5′ side of the template DNAstrand, although the extended strand is not decomposed. This results inthe presence of the DNA strand only on one side.

The aforementioned primer extension product (109 bp primer extensionproduct) was subjected to lambda exonuclease treatment (reaction at 37°C. for 1 hour). The composition of the reaction solution used is shownin Table 4. TABLE 4 Composition of reaction solution 67 mM glycine-KOH(pH 9.3) 2.5 mM MgCl₂ 0.5 U lambda exonuclease (USB)

After the reaction at 37° C. for 1 hour, electrophoresis was carried outin 4% agarose gel. The primer extension product was subjected to lambdaexonuclease treatment and electrophoresis, and as a result, a band wasobserved to have been shifted to around 50 bp (Lane 2 in FIG. 7). Therate of migration of the single-stranded DNA is known to have increased,and this was considered to be the product of interest. Accordingly, itwas confirmed that a larger amount of single-stranded DNA could beobtained by treating the primer extension product with lambdaexonuclease.

EXAMPLE 5 Bst DNA Polymerase Extension Reaction Using TspRI Buffer

1. The Reaction with the Bst DNA Polymerase was Carried Out Using TspRIBuffer.

The LAMP product used as a template was the same as in Example 2. Inthis case, dNTPs having final concentrations of 0, 0.25, 0.5, 1.0, and2.0 mM were used. The composition of the reaction solution used is shownin Table 5. TABLE 5 Composition of reaction solution 20 mM Tris-AcetatepH 7.9 50 mM K-Acetate 10 mM Mg-Acetate 1 mM DTT 0.25-2 mM dNTPs 0.1 UBst DNA polymerase (NEW ENGLAND BioLabs) 5 μM Primer

After the reaction at 37° C. for 1 hour, the reaction product waselectrophoresed in 2% agarose gel and transferred to a nylon membrane(BiodyneB, Pall) in order to confirm the implementation of primerextension. Whether or not this membrane contained the label substance inthe electrophoresis product was detected using the DIG Nucleic AcidDetection Kit (Roche) (FIG. 8). As a result, the occurrence of extensionfrom the primer by the Bst DNA polymerase with the use of TspRI bufferwas confirmed.

This indicates that use of this method can decrease a necessary cost bysharing a buffer. 2. Since the occurrence of extension with the use ofthe TspRI buffer was confirmed, whether or not the reaction subsequentto the LAMP could be simultaneously carried out was then examined. Thecomposition of the reaction solution used is shown in Table 6. TABLE 6Composition of reaction solution 20 mM Tris-Acetate pH 7.9 50 mMK-Acetate 10 mM Mg-Acetate 1 mM DTT 1 mg/ml BSA 0.5 mM dNTPs 7.5 U TspRI(NEW ENGLAND BioLabs) 5 μM Primer

The reaction solution (1/50 amount) after the LAMP reaction was added tothe reaction system and allowed to react at 37° C. for 1 hour. Theamount of the Bst DNA polymerase in the reaction solution was 0.16 U. Itwas confirmed in the Examples that 0.1 U of enzyme is sufficient toproceed the reaction. After the reaction, dot blot hybridization wascarried out using the product as a probe (FIG. 9). The blotted DNAs wereas follows.

1: 82 bp antisense oligo DNA

2: 82 bp sense oligo DNA

3: 109A oligo

4: 109B oligo

As a result, a potent signal was observed at position (1) where the 82bp antisense oligo was blotted. A weak signal observed on the senseoligo side (2) was considered to be generated due to the portion thatremained uncleaved in the reaction by TspRI. Thus, the production ofspecific amplification product was confirmed.

These results indicate that the use of this method can decrease cost aswell as shorten necessary time.

EXAMPLE 6 Insertion of Restriction Enzyme Recognition Sequence UsingLAMP Primer

1. In order to obtain a strand-specific single-strand from a templatecontaining no TspRI site, the LAMP reaction was carried out using thefollowing inner primers, etc. having the TspRI recognition sequenceinserted therein and, as a template, 5 ng of pUC 19 plasmid (SEQ ID NO:19).

Inner primer (1600 nM: the underlined portion indicates the TspRIrecognition sequence-inserted portion)

Forward: 5′-GACCAAGTTTACTCATATATACGACACTGGAGATCCTTTTAAATTAAAAATGAAG-3′(SEQ ID NO: 20)

Reverse: 5′-TGAGGCACCTATCTCAGCGATGACAGTGTCACGGGGAGTCAGGCAACTAT-3′ (SEQID NO: 21)

Outer Primer (400 nM) Forward: 5′-TCAAAAAGGATCTTCACCTA-3′ (SEQ ID NO:22) Reverse: 5′-GTATCGTAGTTATCTACACG-3′ (SEQ ID NO: 23)

Loop Primer (800 nM) Forward: 5′-ACTTTAGATTGATTTAAAA-3′ (SEQ ID NO: 24)Reverse: 5′-TCTGTCTATTTCGTTCATCC-3′ (SEQ ID NO: 25)

The composition of the reaction solution was the same as in Example 1,and the LAMP reaction was carried out at 62.8° C. for 2 hours.Thereafter, the LAMP amplification product was digested with TspRI inthe same manner as in Example 1. The LAMP product and the TspRI digestwere electrophoresed in 2% agarose gel, and as a result, these wereconfirmed to be sizes of interest (FIG. 10).

2. Subsequently, DG-labeling was carried out by primer extension and dotblot hybridization was carried out. The blotted DNAs were as follows.

1: 109A oligo

2: 109B oligo

As a result, it was confirmed that a signal could be generated only atthe spot of interest (FIG. 11).

This indicates that the restriction enzyme recognition sequence can beinserted into a template containing no TspRI site by the LAMP method,and, by using this as a template, a strand-specific single strand can beobtained.

All publications, patents, and patent applications cited herein areincorporated herein by reference in their entirety.

INDUSTRIAL APPLICABILITY

According to the present invention, either one of the sense or antisensestrand of the double-stranded nucleic acid can be selectivelysynthesized. This single-stranded DNA can be used as a probe, etc. forhybridization assay. The present invention provides an efficient andcost-effective method for synthesizing single-stranded DNA.

Free Text of Sequence Listing

SEQ ID NO: 1: T7TspRI primer (Forward)

SEQ ID NO: 2: T7TspRI primer (Reverse)

SEQ ID NO: 3: TspRI-BamHI-TspRI site

SEQ ID NO: 4: Inner primer (Forward)

SEQ ID NO: 5: Inner primer (Reverse)

SEQ ID NO: 6: Outer primer (Forward)

SEQ ID NO: 7: Outer primer Reverse)

SEQ ID NO: 8: Loop primer Forward)

SEQ ID NO: 9: Loop primer Reverse)

SEQ ID NO: 10: pBluescript II

SEQ ID NO: 1: Multiple cloning site from pBluescript II

SEQ ID NO: 12: pUC19 109 bp Sau3AI fragment (sense)

SEQ ID NO: 13: pUC19 82 bp Sau3AI fragment (sense)

SEQ ID NO: 14: pUC19 82 bp Sau3AI fragment (antisense)

SEQ ID NO: 15: 109A oligo: Designed oligonucleotide (sense) based onpUC19 109 bp Sau3AI fragment)

SEQ ID NO: 16: 109B oligo: Designed oligonucleotide (antisense) based onpUC19 109 bp Sau3AI fragment)

SEQ ID NO: 17: Inner primer (Forward)

SEQ ID NO: 18: Inner primer (Reverse)

SEQ ID NO: 19: pUC19

SEQ ID NO: 20: Inner primer (Forward)

SEQ ID NO: 21: Inner primer (Reverse)

SEQ ID NO: 22: Outer primer (Forward)

SEQ ID NO: 23: Outer primer (Reverse)

SEQ ID NO: 24: Loop primer (Forward)

SEQ ID NO: 25: Loop primer (Reverse)

1. A method for synthesizing a single-stranded nucleic acid comprisingthe following steps of: 1) cleaving, with a restriction enzyme,double-stranded DNA having a restriction enzyme recognition sequence ata portion closer to the 5′ side than the target sequence in such amanner that a) a fragment having an overhanging 3′ terminus is formed,and b) base sequences of the single-stranded regions of the fragmentsafter the cleavage differ from each other; 2) to the single-strandedregion of the DNA fragment that was cleaved with the restriction enzyme,annealing a primer having a base sequence complementary to the region onat least its 3′ terminus; and 3) synthesizing a nucleic acid by a stranddisplacement-type polymerase starting from the 3′ terminus of theprimer.
 2. The method according to claim 1, wherein the double-strandedDNA having the restriction enzyme recognition sequence is provided by amethod comprising the following steps of: 1) cloning the DNA to beamplified using a vector having the restriction enzyme recognitionsequence in the cloning site or adjacent to the cloning site; and 2)amplifying the region containing the restriction enzyme recognitionsequence and the DNA to be amplified by the DNA amplification methodusing a primer that anneals on the restriction enzyme recognitionsequence or the 3′ side thereof.
 3. The method according to claim 2,wherein the DNA amplification method is the LAMP method.
 4. The methodaccording to claim 1, wherein the double-stranded DNA having therestriction enzyme recognition sequence is provided by the DNAamplification method using a primer containing the restriction enzymerecognition sequence.
 5. The method according to claim 4, wherein theDNA amplification method is the LAMP method using the primers describedin the following A) and B): when a first arbitrary sequence F1c and asecond arbitrary sequence F2c are selected in that order from the 3′terminus of the target sequence on the first DNA strand of thedouble-stranded DNA toward the 3′ terminus on the DNA strand, and athird arbitrary sequence RI and a fourth arbitrary sequence R2 areselected in that order from the 5′ terminus of the target sequencetoward the 5′ terminus on the DNA strand, A) a primer containingsequence F2, which is complementary to F2c, and the same sequence assequence F1c in that order from the 3′ side toward the 5′ side or aprimer containing sequence F2, which is complementary to F2c, therestriction enzyme recognition sequence, and the same sequence as F1c inthat order from the 3′ side toward the 5′ side; and B) a primercontaining the same sequence as R2, the restriction enzyme recognitionsequence, and sequence R1c, which is complementary to R1, in that orderfrom the 340 side toward the 5′ side.
 6. The method according to claim5, wherein the step of synthesizing DNA comprises the use of an outerprimer that anneals to the portion closer to the 3′ side than the innerprimer.
 7. The method according to any one of claims 1 to 6, wherein thesingle-stranded region at the cleavage site created by the restrictionenzyme comprises at least 5 bases.
 8. The method according to any one ofclaims 1 to 6, wherein the single-stranded region at the cleavage sitecreated by the restriction enzyme comprises at least 7 bases.
 9. Themethod according to any one of claims 1 to 6, wherein the primer isbound to or modified to be bindable to a detectable label substance orsolid phase.
 10. An inner primer pair comprising the following A) andB): when a first arbitrary sequence F1c and a second arbitrary sequenceF2c are selected in that order from the 3′ terminus of the targetsequence on the first DNA strand of the double-stranded DNA toward the3′ terminus on the DNA strand and a third arbitrary sequence R1 and afourth arbitrary sequence R2 are selected in that order from the 5′terminus of the target sequence toward the 5′ terminus on the DNAstrand, A) a primer containing sequence F2, which is complementary toF2c, and the same sequence as F1c in that order from the 3′ side towardthe 5′ side, or a primer containing sequence F2, which is complementaryto F2c, a recognition sequence of the following restriction enzyme, andthe same sequences as sequence F1c in that order from the 3′ side towardthe 5′ side, wherein the restriction enzyme is capable of a) forming afragment having an overhanging 3′ terminus, and b) cleaving so as tomake base sequences of the single-stranded regions of the fragmentsafter the cleavage differ from each other; and B) a primer containingthe same sequence as R2, a recognition sequence of the restrictionenzyme, and sequence R1c, which is complementary to R1, in that orderfrom the 3′ side toward the 5′ side.
 11. A vector for synthesizing asingle-stranded nucleic acid comprising the following base sequence inthe cloning site or adjacent to the cloning site, wherein the basesequence is cleaved with a restriction enzyme in such a manner that a) afragment with an overhanging 3′ terminus is formed, and b) basesequences of the single-stranded regions of the fragments after thecleavage differ from each other.
 12. A reagent kit for synthesizing asingle-stranded nucleic acid comprising at least the followingreagents: 1) a restriction enzyme capable of: a) forming a fragmenthaving an overhanging 3′ terminus, and b) cleaving so as to make thebase sequences of fragments different from each other; 2) a primercapable of annealing to the single-stranded region of the DNA fragmentcleaved with the restriction enzyme; 3) a strand displacement-typepolymerase; and 4) the inner primer according to claim 10 or the vectorfor synthesizing a single-stranded nucleic acid according to claim 11.